Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies have demonstrated that the vitamin pyridoxal phosphate can alter the physicochemical properties of glucocorticoid receptors. We now report the localization of a pyridoxal phosphate binding site within the mero-receptor domain of this glucocorticoid receptor. Mero-glucocorticoid receptors that are generated by trypsin (10 micrograms/ml) or chymotrypsin (100 micrograms/ml) digestion of intact receptors sediment as 2.6 S species on 5-20% sucrose gradients in the presence or absence of pyridoxal phosphate. Mero-glucocorticoid receptors prepared by exogenous proteinases are hydrophobic and show no affinity for DEAE Bio-Gel A. Treating either trypsin-generated or chymotrypsin-generated mero-receptors with pyridoxal phosphate rapidly converts the proteins (60 and 35%, respectively) into forms that bind to DEAE Bio-Gel A. Induction of DEAE binding is specific to pyridoxal phosphate, for treating mero-receptors with pyridoxal, pyridoxamine or pyridoxine phosphate is ineffective. Furthermore, DEAE binding cannot be induced by adding other pyridoxal phosphate-treated cytosols to untreated mero-receptors. High-resolution polyacrylamide gel isoelectric focussing studies indicated that treating mero-receptor generated by either proteinase with pyridoxal phosphate shifted the isoelectric points of both to lower pH values. The conversion of the mero-receptor to a more acidic form also occurred when the intact glucocorticoid receptor was treated with the vitamin prior to proteolysis. These studies localize at least one pyridoxal phosphate binding site on the mero-receptor domain of the rat thymocyte glucocorticoid receptor.
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PMID:Localization of pyridoxal phosphate binding site on the mero-receptor domain of the glucocorticoid receptor. 646 4

Because of the profound importance glucocorticoids have in dermatologic therapy, we studied the glucocorticoid receptor in human skin. A cytosol fraction was prepared from frozen skin by homogenization and centrifugation. When reacted with [3H]dexamethasone, this cytosol contained saturable, low-capacity binding. The glucocorticoid binding was stabilized by a protease inhibitor, phenylmethylsulfonylfluoride, and by sodium molybdate and was destroyed by trypsin. Sedimentation analysis of the glucocorticoid binding protein showed an 8S to 4S transition in high salt, a property of many known steroid hormone receptors. The binding was steroid specific, supporting the conclusion that this binding protein was a glucocorticoid receptor. The receptor molecule had a frictional ratio of 1.60 and a Mr of about 226,000 under low-salt conditions (0.05 M KCl) and a frictional ratio of 1.86 and a Mr of about 100,000 under high-salt conditions (0.3 M KCl) consistent with a nonglobular, elongated molecule. Isoelectric focusing showed that the receptor had 2 molecular species with isoelectric points of approximately 5.8 and 7.5. Quantitation of receptor in human skin showed 4-7 times more receptors in the epidermis and papillary dermis than in the lower dermis and nearly equal numbers in epidermis and papillary dermis. The concentration of receptors varied in different anatomic areas, with male foreskin showing the highest concentration, followed by female face, breast, and abdominal skin. Interestingly, the concentration of glucocorticoid receptors also varied with age; the highest levels were present at the extremes of life and a significantly lower level at midlife.
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PMID:Characterization of the glucocorticoid receptor in human skin. 661 66

Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity.
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PMID:Comparison between different rabbit antisera against the glucocorticoid receptor. 662 Oct 34

The activated glucocorticoid receptor (GR) from rat liver cytosol was purified by sequential chromatography on DNA-cellulose and DEAE-Sepharose. Analysis by sodium dodecyl sulfate-gel electrophoresis demonstrated a main band with Mr = 94,000 (94K band). Two minor bands with Mr = 79,000 (79K band) and 72,000 (72K band) were also seen in this preparation. Photoaffinity labeling showed that the hormone is bound to the 94K and 79K components but not to the 72K component. Immunoblotting using antibodies raised against the 94K protein demonstrated cross-reactivity between the 94K and 79K components but not with the 72K species. The 72K species could be partially separated from the 94K and 79K components by density gradient centrifugation. Limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of a 39K fragment which still retained the hormone and could be bound to DNA-cellulose. The 72K component was not affected by digestion with trypsin or alpha-chymotrypsin. However, chromatography on DNA-cellulose of the alpha-chymotrypsin-treated GR resulted in elution of the 72K component in the flow-through of the column while the 39K fragment was retained on the column and eluted with 0.18 M NaCl. In the control experiment where no alpha-chymotrypsin treatment was performed, the 72K component could not be detected in the flow-through fraction but was eluted together with the 94K and 79K components at 0.18 M NaCl. These results suggest that the 72K protein might be bound to the 94K and/or 79K component. The 39K fragment did not bind antibodies raised against the 94K protein. The 39K fragment was further degraded by trypsin but not by alpha-chymotrypsin to a 27K and a 25K fragment while both still retained the ligand. These data obtained with limited proteolysis of the purified GR are in agreement with previous findings on proteolysis of the GR in crude cytosol (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865; Carlstedt-Duke, J., Okret, S., Wrange, O., and Gustafsson, J.-A. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 4260-4264).
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PMID:Characterization of the purified activated glucocorticoid receptor from rat liver cytosol. 670 18

The in vitro nuclear binding of rat uterine estrogen-receptor complexes has been studied. Heating cytosol from mature rat uterus at 25 C for various times in the presence of 0.15 M KCl resulted in a transient increase in nuclear binding activity, followed by irreversible loss of this activity. The molecular state of these complexes heated at 25 C in the presence of 0.15 M KCl was determined using Sephadex G-200 chromatography and sucrose density centrifugation at high ionic strength (0.4 M KCl). Gel filtration resulted in steroid-binding activity in the void volume. Sucrose density gradient analysis revealed a broad peak, ranging from approximately 5-20S. When cytosol was heated at 25 C in the presence of 10 mM molybdate to block the temperature-induced activation of receptor, nuclear binding ability was easily recovered by dialysis, while heating already activated estrogen receptor in the presence of 0.15 M KCl and 10 mM molybdate caused irreversible loss of nuclear binding ability. When cytosols prepared from immature rats (19-23 days old) were heated at 25 C in the presence of 0.15 M KCl, only a minimum loss of nuclear binding ability was shown. The radioactive peak in a high salt sucrose density gradient appeared almost exclusively in the 5S region. However, the addition of receptor-free mature uterine cytosol to estrogen-receptor complexes from immature rat uterus caused a marked loss of nuclear binding utility, with a resultant receptor aggregation, whereas rat liver cytosol had no effect on this reaction. Furthermore, heating liver glucocorticoid receptor did not cause a loss of nuclear binding ability even in the presence of receptor-free adult rat uterine cytosol. These observations suggest that there is a factor(s) in rat uterus which recognizes only activated estrogen receptor and induces receptor aggregation and a rapid loss of the nuclear binding ability of receptor in a KCl concentration- and temperature-dependent manner. Preliminary characterization indicates that this factor is macromolecular in nature and resistant to RNase and trypsin treatment, but labile at 100 C.
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PMID:A modulator which converts activated estrogen receptor to a biologically inactive aggregated form. 679 27

Activation of the mouse liver glucocorticoid receptor resulted in the generation of a protein of very different characteristics from that found previously in the mouse AtT-20 pituitary tumor cell line [Vedeckis, W. V. (1981) Biochemistry 20, 7237-7245]. Ion-exchange and adsorption chromatography showed that the activated liver receptor was a more basic protein--it eluted earlier from a DEAE-cellulose column, while a later elution was observed upon phosphocellulose and DNA-cellulose chromatography. Further experiments showed that this was due to proteolysis of the liver receptor to a smaller form (3.2 S; Rs = 3.9 nm; Mr = 53 000; f/f0 = 1.45) after activation. Mero-receptor (2.4 S; Rs = 2.4 nm; Mr = 24 000; f/f0 = 1.15) was detectable when cytosol was chromatographed on hydroxylapatite or was treated with trypsin. These proteolytic fragments are similar to those obtained for various other steroid hormone receptors. Mixing experiments with liver cytosol showed that the AtT-20 glucocorticoid receptor could also be cleaved to these fragments. Endogenous proteases are apparently in low concentration in this cell line. Finally, activation appears to result in the exposure of the protease-sensitive regions, since sodium molybdate, which prevents receptor activation, also renders the receptor resistant to proteolysis.
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PMID:Limited proteolysis of the mouse liver glucocorticoid receptor. 684 99

The distribution, metabolism and protein-binding of [3H] corticosterone in the rat liver was studied with respect to time and sex. The maximum recovery from the cell nuclei occurred 5 min after injection of the steroid. At this time, ten times more radioactivity was recovered from the liver cell nuclei of male animals compared to females. The recovered radioactivity in the cell nuclei was identified as mainly unmetabolised corticosterone and 5 alpha-dihydrocorticosterone. The radioactivity bound to the cytosolic glucocorticoid receptor protein also appeared to consist of unmetabolised corticosterone and 5 alpha-dihydrocorticosterone. However, 5 alpha-dihydrocorticosterone was found to have very little biological activity. The glucocorticoid receptor was found to exist in two forms with Stokes radii of 6.1 and 3.6 nm, respectively, following in vivo labelling with [3H] dexamethasone. The 6.1 nm form could be converted into the 3.6 nm form by incubation with trypsin, papain, alpha-chymotrypsin or an extract of purified rat liver lysosomes. Further incubation resulted in an even smaller form with a Stokes radius of 1.9 nm. With the help of biospecific adsorption chromatography based on the differential affinity of the activated and nonactivated complexes for DNA, the 6.1 nm form of the glucocorticoid-receptor complex was purified to near homogeneity by a rapid and simple technique. An immunoglobulin fraction from serum of a rabbit immunized with a highly purified preparation of glucocorticoid receptor contained specific antibodies to the receptor. The antibodies cross-reacted with the glucocorticoid receptor from human normal lymphocytes, chronic lymphatic leukemia cells and human hippocampus. Activated glucocorticoid receptor protein binds selectively in vitro to a cloned fragment of murine mammary tumor virus DNA. In contrast, the receptor fails to bind selectively to DNA restriction fragments from E coli plasmids pBR 322 and RSF 2124 or from bacteriophages alpha and T4.
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PMID:Glucocorticoid-binding proteins in rat liver. 695 93

This paper further characterizes a protein we have demonstrated in Candida albicans which has the ability to bind corticosterone and related steroid hormones. Fungal cells are disrupted and cytosol is incubated with [3H]corticosterone for 3 h at which time peak steady state binding is achieved. Bound hormone is separated from free using Sephadex G-50 minicolumns or dextran-coated charcoal. Binding was found to be a linear function of protein concentration. The bound hormone co-migrates with authentic corticosterone in thin layer chromatographic systems indicating no metabolism of the radioprobe. Scatchard analysis of the binding in the pseudohyphal form of C. albicans yielded values of 6.3 nM for the Kd and a binding capacity of about 650 fmol/mg of cytosol protein; both determinations are comparable to our findings in the yeast form of this organism. A series of sterols were tested for their ability to displace [3H]corticosterone from the yeast binder, and the results show that the binder is remarkably selective and stereo specific. Physical-chemical studies show the binder to be degraded at high temperatures and that binding is destroyed by trypsin and sulfhydryl blockers. The protein sediments at 4 S on sucrose gradients and does not exhibit ionic dependent aggregation. The molecular weight is estimated to be approximately 43,000 daltons by gel chromatography. We hypothesize that this intracellular protein may represent a primitive form of either the mammalian glucocorticoid receptor or the plasma corticosteroid-binding globulin.
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PMID:Characterization of a unique corticosterone-binding protein in Candida albicans. 704 Mar 89

Cytosol glucocorticoid receptor of hamster Malignant Melanoma No. 1 (MM1) was characterized by dextran-coated charcoal and sucrose gradient assays. MM1 contains a specific, saturable, high-affinity (Kd 2.5 nM; 73.3 fmol/mg cytosol protein) receptor for glucocorticoid. The receptor sedimented at 7 to 8S in low-salt buffer and 5.5S in 0.4 M KCl and was sensitive to proteolysis by trypsin. Glucocorticoid receptor was inactivated by 40% (NH4)2SO4. Addition of 20 mM molybdate to the homogenizing buffer prevented inactivation. Adrenalectomy increased MM1 receptor content without altering affinity. Chronic exposure of intact or adrenalectomized hamsters to gluco- and high-dose mineralocorticoid significantly decreased the amount of unbound glucocorticoid receptors available for binding. Exposure of intact hamsters to high doses of mineralocorticoid also decreased apparent receptor affinity. These results suggest that hamster MM1 contains a high-affinity cytosol receptor for glucocorticoids similar to that of other glucocorticoid-responsive tissues, which may mediate the action of adrenal steroids on tumor behavior.
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PMID:Effect of adrenal manipulation on glucocorticoid receptors in MM1 hamster melanoma. 707 6

The glucocorticoid receptor in cytosol from human brain was studied using isoelectric focussing in slabs of polyacrylamide gel. [3H]Dexamethasone was used as tracer for receptor analysis. The glucocorticoid receptor from human brain was compared to the glucocorticoid receptor in rat brain. A similar peak of radioactivity with a pI of about 6.1 was obtained by isoelectric focussing of cytosol from both human brain and rat brain. The trypsin-induced fragmentation patterns of the glucocorticoid receptor from human brain and rat brain were very similar when analyzed by isoelectric focussing. The hormone specificity of the glucocorticoid receptor in human brain and in rat brain cytosol was compared by competition experiments using unlabelled dexamethasone, betamethasone, cortisol and corticosterone as competitors. No difference between human brain and rat brain cytosol was detected. It is concluded that the hormone specificity and the protein structure of the glucocorticoid receptors in human brain and in rat brain are similar.
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PMID:A qualitative comparison of the glucocorticoid receptor in cytosol from human brain and rat brain. 728 14


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